Methods and compositions for the detection and/or quantification of gram positive bacterial contaminants

ABSTRACT

The invention provides methods and compositions for the detection and/or quantification of a Gram positive bacterial contaminant in a sample. In particular, the invention provides hemocyte-based preparations, methods of making such hemocyte-based preparations, and methods of using such hemocyte-based preparations for the detection and/or quantification of the Gram positive bacterial contaminant.

RELATED APPLICATIONS

This application claims the benefit of and priority to U.S. PatentApplication Ser. No. 60/632,785, filed Dec. 2, 2004, the entirety ofwhich is incorporated herein by reference.

GOVERNMENT SUPPORT

This invention was made in part with government support under grantnumber NAG 2-1263 from the National Aeronautics and SpaceAdministration. The United States Government has certain rights in thisinvention.

FIELD OF THE INVENTION

The present invention relates generally to methods and compositions fordetecting and/or quantifying Gram positive bacteria in a sample. Moreparticularly, the invention relates to a hemocyte-based preparationuseful in detecting and/or quantifying Gram positive bacteria in asample, and to methods of making and using such a preparation.

BACKGROUND OF THE INVENTION

Microbial contamination by, for example, Gram positive bacteria, Gramnegative bacteria, yeast, fungi, and molds may cause severe illness and,in some cases, even death in humans. Manufacturers in certainindustries, for example, the pharmaceutical, medical device, water, andfood industries, must meet exacting standards to verify that theirproducts do not contain levels of microbial contaminants that wouldotherwise compromise the health of the recipient. These industriesrequire frequent, accurate, and sensitive testing for the presence ofsuch microbial contaminants to meet certain standards, for example,standards imposed by the United States Food and Drug Administration(USFDA) or the Environmental Protection Agency. By way of example, theUSFDA requires certain manufacturers of pharmaceuticals and invasivemedical devices to establish that their products are free of detectablelevels of Gram negative bacterial endotoxin.

To date, a variety of assays have been developed to detect the presenceand/or amount of a microbial contaminants in a test sample. One familyof assays use hemocyte lysates prepared from the hemolymph ofcrustaceans, for example, horseshoe crabs. These assays typicallyexploit, in one way or another, a clotting cascade that occurs when thehemocyte lysate is exposed to a microbial contaminant. For example, FIG.1 shows a schematic representation of certain clotting cascades known tobe present in hemocyte lysate produced from the hemolymph of thehorseshoe crab, Limulus polyphemus. Such lysates are known in the art asLimulus amebocyte lysate or LAL.

As shown in FIG. 1, the coagulation system of LAL, like the mammalianblood coagulation system, comprises at least two coagulation cascadesthat include an endotoxin or lipopolysaccharide (LPS) mediated pathway(the Factor C pathway) and a (1→3)-β-D glucan mediated pathway (theFactor G pathway). See, for example, Morita et al. (1981) FEBS LETT.129: 318-321; and Iwanaga et al. (1986) J. PROTEIN CHEM. 5: 255-268.

It is understood that Gram negative bacteria can be detected using LALbased assays. For example, Gram negative bacteria produce endotoxin orLPS, which after binding to LPS binding protein activates the Factor Cpathway in LAL (see, FIG. 1). The endotoxin or LPS-mediated activationof LAL is well understood and has been thoroughly documented in the art.See, for example, Levin et al. (1968) THROMB. DIATH. HAEMORRH. 19: 186;Nakamura et al. (1986) EUR. J. BIOCHEM. 154: 511; Muta et al. (1987) J.BIOCHEM. 101: 1321; and Ho et al. (1993) BIOCHEM. & MOL. BIOL. INT. 29:687. When bacterial endotoxin is contacted with LAL, the endotoxininitiates a series of enzymatic reactions, known as the Factor Cpathway, that are understood to involve three serine protease zymogenscalled Factor C, Factor B, and pro-clotting enzyme (see, FIG. 1).Briefly, upon exposure to endotoxin, the endotoxin-sensitive factor,Factor C, is activated. Activated Factor C thereafter hydrolyses andactivates Factor B, whereupon activated Factor B activates proclottingenzyme to produce clotting enzyme. The clotting enzyme thereafterhydrolyzes specific sites, for example, Arg¹⁸-Thr¹⁹ and Arg⁴⁶-Gly⁴⁷ ofcoagulogen, an invertebrate, fibrinogen-like clottable protein, toproduce a coagulin gel. See, for example, U.S. Pat. No. 5,605,806.

Furthermore, it is also understood that (1→3)-β-D glucans and otherLAL-reactive glucans, produced by microorganisms such as yeasts andmolds, can also activate the clotting cascade of LAL, through adifferent enzymatic pathway known as the Factor G pathway (see, FIG. 1).It is understood that Factor G is a serine protease zymogen that becomesactivated by (1→3)-β-D glucan or other LAL reactive glucans. Uponexposure to (1→3)-β-D glucan, for example, Factor G is activated toproduce activated Factor G. It is understood that activated Factor Gthereafter converts the proclotting enzyme into clotting enzyme,whereupon the clotting enzyme converts coagulogen into coagulin.

Presently, LAL is employed as the amebocyte lysate of choice in manyassays for detecting the presence of Gram negative bacteria, fungus ormolds because of its sensitivity, specificity, and relative ease foravoiding interference by other components that may be present in asample. For example, LAL, when combined with a sample containingbacterial endotoxin and optionally with certain LAL substrates, reactswith the endotoxin in the sample to produce a detectable product, suchas a gel, increase in turbidity, or a colored or light-emitting productin the case of a synthetic chromogenic substrate. The resulting productmay be detected, for example, either visually or by the use of anoptical detector.

In contrast, assays of comparable sensitivity and specificity fordetecting and/or quantifying Gram positive bacterial contaminants havebeen more difficult to develop. One assay for detecting Gram positivebacterial contamination, known as the In Vitro Pyrogen Test (IPT), isavailable from Charles River Laboratories (Wilmington, Mass.). The IPTassay is an in vitro alternative to the rabbit pyrogen test, and is anELISA-based assay that uses fresh or cryoproserved human whole blood.When exposed to pyrogens, immune cells within the whole blood produceinterleukin-1β that is detected in the ELISA assay. The IPT assay,however, does not selectively detect the presence of Gram positivebacteria, as it is activated by pyrogens present in Gram positivebacteria, Gram negative bacteria, yeasts and viruses.

Although the detection of bacterial, yeast and fungal contamination canbe extremely important, the ability to discriminate between thesedifferent organisms can provide useful information about an infectiousagent causing an infection in an individual or the source and type ofcontamination present in a test sample. For example, once an infectiousagent has been identified, a physician can then prescribe the mostappropriate medication for treating an infection. Furthermore, once thetype of bacterial, yeast or fungal contamination has been identified,then this type of information may speed up the process of identifyingthe source of contamination in, for example, a water supply. As aresult, once the source of contamination has been identified, furthercontamination can be mitigated. Although methods and compositionscurrently are available for specifically detecting Gram negativebacteria, yeasts, and molds, there is still an ongoing need for furthermethods and compositions for specifically detecting Gram positivebacteria in a sample of interest.

SUMMARY OF THE INVENTION

The invention generally provides methods and compositions useful indetecting the presence and/or amount of lipoteichoic acid, a moleculefound within the cell wall of Gram positive bacteria. This informationcan be used to determine whether Gram positive bacteria are present in atest sample and also can be used to measure the extent of contaminationin a test sample.

In one aspect, the invention provides a method of producing apreparation for detecting the presence and/or amount of lipoteichoicacid in a sample. The invention comprises the steps of: (a) providing ahemocyte preparation harvested from a crustacean selected from a groupconsisting of Cancer borealis, Cancer irroratus, Hemigrapsus sanguineus,and Limulus polyphemus, wherein the hemocytes contain lipoteichoic acidreactive material; (b) releasing the lipoteichoic acid reactive materialfrom the hemocytes; and (c) harvesting the lipoteichoic acid reactivematerial released from the hemocytes.

In step (b), the lipoteichoic acid can be released from the hemocytes bya variety of procedures. For example, the hemocytes may be lysed byconventional procedures, for example, by osmotic shock, sonication,homogenization, and ultracentrifugation. Alternatively, the outermembrane of the hemocytes may be rendered permeable, for example,selectively permeable, to the lipoteichoic acid reactive materialdisposed within the hemocytes. In this approach, the hemocytes can berendered permeable to lipoteichoic acid reactive material by exposingthe hemocytes to a membrane permeabilizing agent. Although a variety ofmembrane permeabilizing agents can be used, a preferred membranepermeabilizing agent is an ionophore, for example, a calcium ionophore.The ionophore is admixed with the preparation to give a finalconcentration in the range from about 0.1 μM to about 100 μM, forexample, from about 1 μM to about 10 μM. When a calcium ionophore isused, the ionophore preferably is combined with divalent cations, forexample, calcium ions.

The resulting preparation of lipoteichoic acid reactive materialcomprises the pro-enzyme pro-phenol oxidase. Furthermore, thepreparation, when made, contains lipoteichoic acid reactive materialthat is substantially inactive at the time the preparation is made.However, when the preparation is later combined with lipoteichoic acid,the lipoteichoic acid reactive material becomes activated whereupon thepro-phenol oxidase is converted into phenol oxidase. In order to assaythe amount of phenol oxidase in a test sample, the preparation can becombined with one or more substrates for phenol oxidase. The phenoloxidase then acts on or converts the substrate into a product that canbe detected and/or quantified either by visual inspection or by means ofa detector, for example, an optical detector, such as, aspectrophotometer, fluorimeter, or the like.

In another aspect, the invention provides preparations of lipoteichoicacid reactive material that can be produced by the foregoing methods.The preparations comprise the pro-enzyme pro-phenol oxidase. Whenproduced, the lipoteichoic acid reactive material contained within thepreparation is substantially inactive. However, when exposed tolipoteichoic acid, for example, lipoteichoic acid present in and on Grampositive bacteria, the lipoteichoic acid reactive material becomesactivated and the pro-phenol oxidase is converted into phenol oxidase.The active phenol oxidase enzyme can then act on or convert a substrateto produce a detectable product (either visually detectable or by meansof a detector).

The invention provides a composition comprising an isolated enzymepreparation derived from crustacean hemocytes. The preparation comprisespro-phenol oxidase, which becomes converted into phenol oxidase when theenzyme preparation is contacted with lipoteichoic acid, for example,lipoteichoic acid present in or on Gram positive bacteria. It isunderstood that this type of preparation can be produced by renderingthe outer membranes of crustacean hemocytes permeable, for example,selectively permeable, to pro-phenol oxidase as well as other componentsof the crustacean's pro-phenol oxidase cascade. Such a preparation canbe produced from the hemocytes derived from crustaceans selected fromthe group consisting of crustaceans selected from the group consistingof Cancer borealis, Cancer irroratus, Carcinus maenas, Hemigrapsussanguineus, and Limulus polyphemus.

The invention also provides a crustacean hemocyte lysate comprisingpro-phenol oxidase, which is converted into phenol oxidase when thehemocyte lysate, after preparation, is subsequently contacted withlipoteichoic acid, for example, lipoteichoic acid present in or on Grampositive bacteria. The crustacean hemocytes can be derived from acrustacean selected from the group consisting of Cancer borealis, Cancerirroratus, Carcinus maenus, Hemigrapsus sanguineas, and Limuluspolyphemus.

The foregoing preparations can be combined with a substrate for phenoloxidase. Accordingly, when the preparation is activated by exposure tolipoteichoic acid reactive material from Gram positive bacteria, thesubstrate is converted into a product that can be detected and/orquantified by visual inspection or by means of a detector, for example,an optical detector.

Depending upon the choice of the starting materials and the preparationconditions, it is possible to produce an isolated enzyme preparationsubstantially free of Factor C activity and/or Factor G activity. Theresulting preparation can remain substantially inactive in the presenceof (i) one of (1→3)-β-D glucan, lipopolysaccharide or proteoglycan, (ii)a combination of (1→3)-β-D glucan and lipopolysaccharide, a combinationof (1→3)-β-D glucan and proteoglycan, or a combination oflipopolysaccharide and proteoglycan, or (iii) a combination of (1→3)-β-Dglucan, lipopolysaccharide and proteoglycan. For example, an enzymepreparation can be produced by permeabilization of the hemocytemembranes of the crustacean Cancer borealis. For example, when hemocytesof the crustacean, Cancer borealis, are rendered permeable tolipoteichoic acid reactive material by exposure to a membranepermeabilizing amount of an ionophore, for example, a calcium ionophore,the resulting preparation containing lipoteichoic reactive material issubstantially free of the Factor C cascade and of the Factor G cascade.As a result, this preparation is activated by, and thus detects thepresence of, Gram positive bacteria but not Gram negative bacteria oryeast and fungus.

In contrast, when hemocytes of the crustacean Limulus polyphemus arelysed by conventional procedures, for example, by osmotic shock,sonication, homogenization, and ultracentrifugation, or renderedpermeable, for example, using a membrane permeabilizing amount of anionophore, the resulting preparation containing lipoteichoic acidreactive material also contains a complete Factor C cascade and a FactorG cascade. As a result, the resulting lysate is activated by, and thusdetects the presence of, Gram positive bacteria, Gram negative bacteria,and yeast and fungus.

In another aspect, the invention provides methods of detecting thepresence and/or amount of Gram positive bacteria in a test sample. Themethod comprises contacting the sample to be tested with one or more ofthe foregoing preparations. It is contemplated that the lysates may beused in a variety of different assays, for example, endpoint chromogenicassays, single-step kinetic assays, and multi-step kinetic assays.Furthermore, it is contemplated that the assays can be run in a varietyof different formats, for example, in a well, a microtitre plate, or inan optical cartridge. The assays can be used to detect the presenceand/or quantity of Gram positive bacteria in a liquid sample ofinterest. In addition, solid samples can be analyzed after contact orsolubilization in a liquid sample. Furthermore, swabs of a solid surfacecan be analyzed after the swab has been contacted or solubilized in aliquid sample.

These aspects and features of the invention may be more completelyunderstood by reference to the drawings, detailed description, andclaims that follow.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention may be more clearly understood by reference to thedrawings, wherein:

FIG. 1 is a schematic representation of the Factor C and Factor Gcascades present in Limulus amebocyte lysate;

FIG. 2 is a schematic representation showing the pro-phenol oxidaseactivation cascades in Cancer borealis hemocytes;

FIG. 3 is a schematic representation showing the pro-phenol oxidaseactivation cascade present in Limulus polyphemus hemocytes;

FIGS. 4A-4D are schematic illustrations of an exemplary cartridge usefulin practicing methods of the invention in perspective view (FIG. 4A),top view (FIG. 4B), side view (FIG. 4C), and end view (FIG. 4D);

FIGS. 5A-5D are schematic illustrations of an exemplary cartridge inwhich FIG. 5A is a view of a bottom half of an exemplary cartridgeshowing the locations of immobilized hemocyte preparation and pro-phenoloxidase substrate, FIG. 5B is a view of a top half of an exemplarycartridge of the invention, FIG. 5C is a cross-sectional view of thefabricated cartridge through section A-A, and FIG. 5D is across-sectional view of the fabricated cartridge through section B-B;

FIG. 6 is a flow chart for an exemplary multi-step kinetic chromogenicassay;

FIG. 7 is a graph showing pro-phenol oxidase reactivity in a Cancerborealis hemocyte preparation in the presence of lipoteichoic acid,peptidoglycan, glucan and lipopolysaccharide;

FIG. 8 is a graph showing the pro-phenol oxidase reactivity of Cancerborealis hemocyte preparation in the presence of Staphylococcusepidermidis (Gram positive) bacterial cells;

FIG. 9 is a graph showing the pro-phenol oxidase reactivity of Cancerborealis hemocyte preparation in the presence of Bacillus subtilis (Grampositive) bacterial cells;

FIG. 10 is a graph showing the change in optical density over time in acartridge-based two-step kinetic assay when a Cancer borealis hemocytepreparation is incubated with different concentrations of lipoteichoicacid; and

FIG. 11 is a graph showing the pro-phenol oxidase reactivity of a Cancerborealis hemocyte preparation combined with crude Limulus amebocytelysate in the presence of peptidoglycan, glucan, lipopolysaccharide andlipoteichoic acid; and

FIG. 12 is a graph showing the pro-phenol oxidase reactivity of Cancerborealis preparation in a multi-step kinetic assay using a fluorescentphenol oxidase substrate in the presence or absence of an esterase.

DETAILED DESCRIPTION

The invention is based, in part, upon the discovery that it is possibleto produce a hemocyte-based preparation that reacts with thelipoteichoic acid present in or on Gram positive bacteria. As a result,the hemocyte preparations of the invention can be used to detect and/orquantitate the presence of Gram positive bacteria in a test sample.

The hemocyte preparations of the invention contain lipoteichoic acidreactive material. Depending upon the source of the starting materialand the hemocyte treatment conditions, it is possible to produce apreparation that reacts specifically with lipoteichoic acid. Thispreparation can detect the presence and/or amount of Gram positivebacteria in a sample of interest. Alternatively, using a differentstarting material and different hemocyte treatment conditions, it ispossible to produce a preparation that reacts with lipoteichoic acid,proteoglycan, lipopolysaccharide and (1→3)-β-D glycan. Accordingly, thispreparation can detect the presence and/or amount of Gram positivebacteria, Gram negative bacteria, and yeast and molds in a sample ofinterest.

FIG. 2 shows the clotting cascades present in hemocytes derived fromCancer borealis. According to FIG. 2, lipoteichoic acid (LTA), whenpresent, binds to a LTA binding protein. Without wishing to be bound bytheory, it is understood that the LTA binding protein then activates aserine protease cascade, which in turn converts a pro-phenol oxidaseactivating enzyme precursor into active pro-phenol oxidase activatingenzyme. The pro-phenol oxidase activating enzyme, when activated,converts pro-phenol oxidase into phenol oxidase. Activated phenoloxidase then reacts with a phenol oxidase substrate, for example,isoprenaline sulfate, to produce a product, for example, a coloredproduct.

FIG. 3 shows the clotting cascades present in hemocytes derived fromLimulus polyphemus. Limulus hemocytes are understood to be reactive withlipoteichoic acid, peptidoglycan, (1→3)-β-D glucan, andlipopolysaccharide, which, by a variety of mechanisms, can producephenol oxidase, which can then react with a phenol oxidase substrate,for example, isoprenaline sulfate, to produce a colored product. Withoutwishing to be bound by theory, it is understood that LTA is bound by aLTA binding protein, proteoglycan is bound by a proteoglycan bindingprotein, (1→3)-β-D glucan is bound by a (1→3)-β-D glucan bindingprotein, and lipopolysaccharide is bound by a lipopolysaccharide bindingprotein. The binding proteins, once bound to their particular ligands,activate one or more serine protease cascades. The serine proteasecascade(s) then converts a pro-phenol oxidase activating enzymeprecursor into a phenol oxidase activating enzyme. The phenol oxidaseactivating enzyme then converts pro-phenol oxidase into phenol oxidase,which then reacts with a phenol oxidase substrate isoprenaline sulfateto produce a product, for example, a colored product.

Accordingly, depending upon the starting material and the extractionprocedure, it is possible to produce a hemocyte preparation that isactivated (i) only by lipoteichoic acid or (ii) by lipoteichoic acid aswell as lipopolysaccharide and (1→3)-β-D glucan. For example, hemocytesmay be harvested from Cancer borealis, which once harvested can eitherbe permeablized or lysed to release the lipoteichoic acid reactivematerial disposed therein.

The term “lipoteichoic acid reactive material” is understood to mean anisolated preparation derived from hemocytes, which, when contacted withlipoteichoic acid is capable of converting pro-phenol oxidase intophenol oxidase.

The source of hemocytes, extraction procedures, assays and assay formatsuseful in the practice of the invention will now be discussed in moredetail below.

Hemocyte Preparation

As discussed, the hemocytes can be harvested from a variety of differentcrustaceans selected from crabs belonging to the Cancer genus, forexample, Cancer borealis, Cancer irratus, Carcinus maenas, Hemigrapsussanguineus (Japanese Shore Crab), crabs belonging to the Limulus genus,for example, Limulus polyphemus, crabs belonging to the Tachypleusgenus, for example, Tachypleus gigas, for example, Tachypleustridentatus, and crabs belonging to the Carcinoscorpius genus, forexample, Carcinoscorpius rotundicauda.

Once harvested, the hemocytes can be lysed using conventional techniquesknown in the art, for example, by osmotic shock, homogenization,ultrasonication and ultracentrifugation. For example, crude lysates maybe produced using the procedure as originally described in Levin et al.(1968) THROMB. DIATH. HAEMORRH. 19: 186, with modification, or in Prior(1990) “Clinical Applications of the Limulus Amebocyte Lysate Test” CRCPress 28-36 and 159-166, and in U.S. Pat. No. 4,322,217.

Alternatively, the outer membranes of the hemocyte can be renderedpermeable to lipoteichoic acid reactive material disposed therein. Thiscan be accomplished using a variety of membrane permeabilizing agentsknown in the art, for example, salt solutions, detergents, antibiotics,and ionophores.

Exemplary salt solutions include, for example, sodium chloride,potassium chloride, sodium acetate, magnesium chloride, magnesiumsulfate, calcium chloride, calcium sulfate, and combinations of salts,such as those found in seawater. Exemplary detergents include, forexample, Triton X-100, Tween-20, Nonidet P-40 and other non-ionicdetergents. Exemplary antibiotics include, for example, gramicidin,polymyxin, and tetracycline. Exemplary ionophores include, for example,calcium ionophores, for example, calcium ionophore A23187, also known asC-7522, available from Sigma Chemical Co., St. Louis, Mo., calciumionophore II 21193 from Fluka, Switzerland, and calcium ionophore IV21198 from Fluka, Switzerland.

The calcium ionophores preferably are admixed with the hemocytepreparation to give a final concentration in the range from about 0.1 μMto about 100 μM, preferably in the range from about 1 μM to about 10 μM.In order to improve the activity of the calcium ionophore, the ionophorecan be combined with divalent cations, for example, calcium chloride inthe range from about 0.1 mM to about 100 mM, preferably in the rangefrom about 1 mM to about 10 mM.

For example, the hemocytes, when harvested, are washed in a saltsolution containing, for example, 0.45 M sodium chloride, 0.1 M glucose,0.1 M cacodylic acid pH 7.0+/−1.0 pH units. The washed hemocytes thenare combined with a release solution containing a ionophore to releasethe lipoteichoic acid reactive material from the hemocytes. An exemplaryrelease solution contains, for example, 0.45 M sodium chloride, 0.1 Mglucose, 0.1 M cacodylic acid, 3 μM calcium ionophore, 5 μM calciumchloride pH 7.0+/−1.0 pH units.

The lipoteichoic acid reactive material, once released from thehemocytes, can be used immediately or stored for later use. For example,the hemocyte preparation may be lyophilized under standard conditions,or frozen and stored at a temperature from −20° C. to −80° C. until use.

The resulting hemocyte preparations contain lipoteichoic acid reactivematerial, which when activated converts pro-phenol oxidase into phenoloxidase. The preparations can also be combined with a phenol oxidasesubstrate including, for example, isoprenaline sulfate (I0261, TCI-GR,Tokyo, Japan), isoproternol hemisulfate salt (I-5752, Sigma ChemicalCo., St. Louis, Mo.), catechol (C-9510, Sigma Chemical Co., St. Louis,Mo.), 4-methylcatechol (M3,420-0 Aldrich Chemical Company Inc.,Milwaukee, Wis.), L-dopa (D-9628, Sigma Chemical Co., St. Louis, Mo.),dopamine (H-8502, Sigma Chemical Co., St. Louis, Mo.), and2′,7′-dichlorodihydrofluorescein diacetate (D-399, Molecular probes,Eugene, Oreg.).

Depending upon the source of the starting material, for example,hemocytes harvested from the crustaceans Cancer borealis, it is possibleto produce a preparation that is substantially free of Factor C activityand/or factor G activity. In other words, these preparations are notactivated by lipopolysaccharide and/or (1→3)β-D glucan, respectively.Similarly, it is possible to produce hemocyte preparations that are notactivated by proteoglycan.

In addition, it is possible to reconstitute a hemocyte preparationhaving a desired specificity for lipoteichoic acid, (1→3) β-D glucan,and lipopolysaccharide. For example, a lipoteichoic acid specifichemocyte preparation harvested from Cancer borealis, can be combinedwith a Factor C specific hemocyte preparation known in the art or aFactor G specific hemocyte preparation known in the art. As such it ispossible to create a hemocyte preparation that is reactive withlipoteichoic acid and either (1→3)β-D glucan or lipopolysaccharide.Alternatively, it is possible to create a hemocyte preparation that isreactive with lipoteichoic acid, (1→3)β-D glucan and lipopolysaccharideby combining a lipoteichoic acid specific hemocyte preparation harvestedfrom Cancer borealis with crude Limulus amebocyte lysate containing boththe Factor C and Factor G cascades. The resulting hemocyte preparation,therefore, is reactive with lipoteichoic acid, (1→3)-β-D glucan andlipopolysaccharide.

In addition, it is understood that under certain circumstances, it ispossible to create a crude Limulus amebocyte lysate that is reactivewith lipoteichoic acid, (1→3)-β-D glucan, lipopolysaccharide andproteoglycan. It appears that crude Limulus amebocyte lysate contains aninhibitor that reduces lipoteichoic acid reactivity. However, thepreparation can be diluted to dilute the concentration of the inhibitorpresent in the hemocyte preparation. Accordingly, by choosing theappropriate dilution, it is possible to produce a crude Limulusamebocyte lysate that is reactive with lipoteichoic acid (present in oron Gram positive bacteria), lipopolysaccharide (present in or on Gramnegative bacteria), (1→3)-β-D glucan (present in or on fungus and molds)and, optionally, proteoglycan (present in or on Gram positive bacteria).

As will be apparent to one of ordinary skill, any buffer and salt knownto promote activation of phenol oxidase in hemocyte preparations, aswell as buffers to avoid extremes of pH that could inactivate thecascade, preferably are included in the hemocyte preparation. Althoughany of the buffers and salts that are understood in the art to becompatible with the hemocyte preparation may be used, buffer solutionscomprising, for example, cacodylic acid, citrate, phosphate, PIPES,MOPS, HEPES, and Tris(tris(hydroxy)aminomethane). Typical formulationadditives may include, without limitation, about 100-300 mM NaCl, about10-100 mM divalent cations (e.g., Mg²⁺ or Ca²⁺), biocompatible buffers,e.g., Tris(tris(hydroxy)aminomethane), to give a final pH of about 6.5to about 8.5, and, if the lysate is to be freeze dried, then sugars,e.g., mannitol or dextran and viscosity increasing agents such aspolyvinyl alcohol (or anti-frothing agents including polyvinyl alcoholand polypropylene glycol).

Exemplary Assays

Assays specific for detecting the presence of Gram positive bacteria ina test sample can be prepared with the hemocyte-based preparations ofthe invention. Although various types of assays can be performed withthe preparation, as discussed below, each of the assays of the inventiongenerally involve contacting the preparation with a test sample in thepresence of a phenol oxidase substrate. Phenol oxidase substratesinclude, for example, isoprenaline sulfate (I0261, TCI-GR, Tokyo,Japan), isoproternol hemisulfate salt (I-5752, Sigma Chemical Co., St.Louis, Mo.), catechol (C-9510, Sigma Chemical Co., St. Louis, Mo.),4-methylcatechol (M3,420-0 Aldrich Chemical Company Inc., Milwaukee,Wis.), L-dopa (D-9628, Sigma Chemical Co., St. Louis, Mo.), dopamine(H-8502, Sigma Chemical Co., St. Louis, Mo.), and2′,7′-dichlorodihydrofluorescein diacetate (D-399, Molecular probes,Eugene, Oreg.).

Inhibition or enhancement of the assay occurs when substances in thetest sample interfere with the hemocyte preparation reaction. Inhibitionresults in a longer reaction time, indicating lower levels of microbialcontamination than may actually be present in the test sample.Enhancement results in shorter reaction time, indicating higher levelsof microbial contamination than may actually be present in the testsample. To verify the lack of inhibition or enhancement, an aliquot oftest sample (or a dilution of the test sample) is “spiked” with a knownamount of an agent representative of the microbial contaminant to bemeasured, for example, lipoteichoic acid. It is recommended that themicrobial contaminant spike results in a final microbial contaminantconcentration in the sample equal to the mid-point, on a log basis,between the microbial contaminant concentration of the highest andlowest standards in the standard curve. For example, in an assay with astandard curve spanning from 50 PPO Units/mL to 0.005 PPO Units/mL,samples should be spiked to contain a final microbial contaminantconcentration of 0.5 PPO Units/mL. In an assay with a standard curvespanning from 1 PPO Units/mL to 0.01 PPO Units/mL, the microbialcontaminant spike should result in a final microbial contaminantconcentration of 0.1 PPO Units/mL.

The spiked sample is assayed in parallel with the unspiked sample. Theresulting microbial contaminant concentration in the unspiked sample andthe microbial contaminant recovered in the spiked sample then arecalculated. The microbial contaminant recovered should equal the knownconcentration of the spike within about 25%. If the test sample (ordilution) is found to inhibit or enhance the reaction, the sample mayrequire further dilution until the inhibition or enhancement isovercome. Initially, one may want to screen for inhibition orenhancement by testing 10-fold dilutions of test sample. Once theapproximate non-inhibitory or non-enhancing dilution is determined, theexact dilution can be found by testing two-fold dilutions around thisdilution. The degree of inhibition or enhancement will be dependent uponthe concentration of the test sample. If several concentrations of thesame sample are to be assayed, it is necessary to establish performancecharacteristics for each concentration independently.

Gram positive bacteria can be detected using the preparations of theinvention in a variety of different assays. For example, end pointchromogenic assays, single-step kinetic assays, and/or multi-stepkinetic assays can be performed in optical cuvettes, microtiter platesand the like. A variety of different assays and assay formats aredescribed, for example, in published U.S. patent application2004-0241788. By way of example, cartridge-based assays are described inmore detail below.

The Cartridge

Exemplary cartridges useful in the practice of the invention are shownschematically in FIGS. 4A-4D. In general, the cartridge is an opticalcartridge containing an immobilized hemocyte preparation for use inhemocyte preparation-based assays. These cartridges may be used alone ortogether with an optical detector, for example, a hand held opticaldetector. Although methods of the invention can be practiced without useof the cartridge, methods of the invention are particularly effectivewhen combined the cartridge to provide a system that can be used in thefield to provide rapid test results. This facilitates quickerelimination and/or treatment of microbial contamination.

A number of assays for the detection and/or quantification of amicrobial contaminant can be performed in the cartridge of theinvention, for example, as illustrated in FIG. 4. The cartridge may beused on its own and the test result detected by eye or it may be used incombination with an optical detector, for example, a hand-held opticaldetector as shown and described in U.S. Pat. No. Des. 390,661.

By way of example and as illustrated in FIGS. 4A-4D, cartridge 1 has asubstantially planar housing fabricated, for example, from a moldablebiocompatible material. The housing may be fabricated from any material,however, transparent and/or translucent glass or polymers are preferred.Preferred polymers include, for example, polystyrene, polycarbonate,acrylic, polyester, optical grade polymers, or any plastic such that theoptical cell is substantially transparent. The housing contains at leastone fluid inlet port 4, at least one optical cell 6, and at least oneconduit 8 having a fluid contacting surface for providing fluid flowcommunication between the fluid inlet port 4 and optical cell 6. Theonly requirements for the optical cell 6 are that it defines a voidcapable of containing a sample to be tested and that a portion of theoptical cell 6 is transparent to light. Cartridge 1 may also have atleast one pump port 12 in fluid flow communication with fluid inlet port4 and optical cell 6 for attaching the cartridge 1 to a pump. The pumpmay then impart a negative pressure via pump port 12 to pull the samplefrom fluid inlet port 4 to optical cell 6. A hemocyte preparation isdisposed on a first region 14 of the fluid contacting surface of conduit8, so that when a sample is applied to fluid inlet port 4, the sampletraverses region 14 and solubilizes or reconstitutes the hemocytepreparation into the sample as it moves toward optical cell 6. This typeof cartridge 1 may be used for determining whether a detectable changehas occurred in the sample. In one embodiment, a phenol oxidasesubstrate is applied to the surface of the conduit 8 at first region 14together with the hemocyte preparation. This type of cartridge 1 may beused for performing, for example, a kinetic chromogenic assay.

In another embodiment, as illustrated in FIGS. 4A-4D, a second region 16of the fluid contacting surface of conduit 8 is spaced apart from anddownstream of first region 14. In this configuration, a hemocytepreparation is disposed at first region 14 and a phenol oxidasesubstrate is disposed at second region 16, so that after the sample iscontacted with the hemocyte preparation in region 14, thesample-hemocyte preparation mixture traverses conduit 8 and contacts thephenol oxidase substrate in region 16. The sample-hemocytepreparation-substrate mixture then traverses conduit 8 to optical cell6. This type of cartridge may be used for performing, for example, anendpoint chromogenic assay or a multi-step kinetic chromogenic assay, asdiscussed in more detail below.

Depending upon the type of assay to be performed, a pre-selected amountof an agent representative of a microbial contaminant, or “spike,” suchas lipoteichoic acid, is disposed on first region 14 of the fluidcontacting surface of one or more conduits 8. Alternatively, the spikemay be disposed on a different region of the conduit 8.

The cartridges can be designed and used according to the type and/ornumber of tests required. For example, a single sample may be tested,for example, in duplicate or triplicate, for example, for researchlaboratory uses or for medical device and biopharmaceutical testing.Alternatively, two or more different samples may be tested individually,for example, for dialysis facility testing of water and dialysate. Thecartridge preferably is a single-use, disposable cartridge that isdiscarded after one use. The cartridge can use approximately 20-100 foldless hemocyte preparation per sample than is used in the conventionalendpoint chromogenic or kinetic chromogenic assays performed inmulti-well plates, and thus provides a less costly andenvironmentally-friendly test. Once a particular assay format has beenchosen, the cartridge may be fabricated as discussed below. All thereagents and materials used to prepare the cartridge preferably are freeof the microbial contaminant for which the cartridge ultimately will beused to test. It is contemplated that the cartridge may be fabricatedwith any hemocyte preparation of choice.

Cartridge Fabrication

In fabricating an exemplary cartridge, it is helpful to combine thehemocyte preparation and phenol oxidase substrate with at least oneresolubilizing agent, such as a sugar or salt, and at least oneanti-flaking agent, such as a polymer, prior to drying the hemocytepreparation onto the solid support.

The resolubilizing agent preferably stabilizes the hemocyte preparationin the dried form and facilitates resolubilization of the reagentsduring the assay. Useful resolubilizing agents include, for example,mannitol, mannose, sorbitol, trehalose, maltose, dextrose, sucrose, andother monosaccharides and disaccharides. The hemocyte preparation andphenol oxidase substrate preferably contain from about 0.01% (w/v) toabout 20% (w/v), more preferably from about 0.1% (w/v) to about 1.0%(w/v) of the resolubilizing agent prior to drying.

The anti-flaking agent is an agent that prevents or reduces thelikelihood that the hemocyte preparation and/or phenol oxidase substratebecomes disassociated from a solid support in the form of a dry flake.The anti-flaking agent preferably also stabilizes the hemocytepreparation or phenol oxidase substrate in the dried form. Usefulanti-flaking agents include, for example, one or more polymers,including, for example, polyethylene glycol, polyvinyl pyrolidone,dextrans, mannitol, and proteins, for example, serum albumin, hemolymphor hemocyanin. The lysate preferably contains from about 0.01% (w/v) toabout 25% (w/v), more preferably from about 0.1% (w/v) to about 1.0%(w/v) of anti-flaking agent prior to drying.

In addition, it has been found that certain polymers reduce theformation of air bubbles (e.g., frothing) when the hemocyte preparationand/or phenol oxidase substrate are resolubilized. Useful anti-frothingagents include polyvinyl alcohol and polypropylene glycol. In order toreduce frothing, the hemocyte preparation and/or phenol oxidasesubstrate may contain from about 0.01% (w/v) to about 10% (w/v), morepreferably from about 0.1% (w/v) to about 1.0% (w/v) anti-frothing agentprior to drying.

An exemplary fabrication process for the cartridge is described withreference to FIG. 5, in which FIG. 5A represents a bottom half 2 ofcartridge 1 and FIG. 5B represents a top half 3 of cartridge 1. Onceprepared, the two halves of the cartridge 1 are joined to one another byadhesive, solvent bonding, ultrasonic welding, snap fit joints, or thelike.

In FIG. 5A, the bottom half 2 of the cartridge 1 defines one half ofeach conduit 8′ (each having a first region 14′ and a second region16′). During fabrication of the bottom half 2 of the cartridge 1, ahemocyte preparation is applied to each first region 14′ and chromogenicsubstrate is applied to each second region 16′. In FIG. 5B, the top half3 of the cartridge 1 defines one half of each conduit 8″. Duringfabrication of top half 3 of the cartridge 1, an agent representative ofa microbial contaminant (i.e., a spike), for example, a preselectedamount of lipoteichoic acid, can, depending on the assay, be applied toregion 14″. Once the reagents have been applied to the respective top 3and bottom 2 halves of the cartridge 1, the cartridge halves 2 and 3then are dried under conditions that preserve the activity of thehemocyte preparation and permit reconstitution of the hemocytepreparation to produce an active hemocyte preparation. In order topreserve the activity of the reagents during drying, the cartridgehalves 2 and 3 are placed in an environment having a temperature fromabout 4° C. to about 40° C., more preferably, from about 10° C. to about35° C., more preferably, from about 15° C. to about 30° C., and arelative humidity from about 0% to about 30%, more preferably, fromabout 2% to about 20%, more preferably from about 4% to about 10%.Drying conditions may include, for example, a temperature of about 25°C. and a relative humidity of about 5%. In an alternative approach, thehemocyte preparation may be dried via freeze drying under standardconditions, about −30° C. to about −40° C. under vacuum.

After drying, the two cartridge halves 2 and 3 are joined to one anotherto create an intact cartridge 1. FIG. 5C is a cross-sectional viewthrough Section A-A′ in which the two halves of the conduit (namely 8′and 8″) together create an intact conduit 8, wherein region 14′ of thebottom 8′ of each conduit contains immobilized hemocyte preparation 20and region 14″ of the top 8″ of one conduit contains immobilizedlipoteichoic acid 22. FIG. 5D is a cross-sectional view through SectionB-B′ in which region 16′ of the bottom 8′ of each conduit containsimmobilized phenol oxidase substrate 24.

The dimensions of a particular cartridge 1 may vary depending upon thenumber and/or type of assays to be performed. However, in oneembodiment, as shown schematically in FIG. 4A, for example, thecartridge 1 has a length of about 10.16 cm (4.00″), width of about 2.54cm (1.00″), and a height of about 0.476 cm (0.188″). The bore of theconduit 8 running from the fluid inlet port 4 to the optical cell 6 isabout 0.127 cm (0.050″), where the hemocyte preparation is dried on aregion 14 of the conduit 8 about 2.381 cm (0.938″) from the fluid inletport 4, and a phenol oxidase substrate is dried on a region 16 of theconduit 8 about 4.65 cm (1.831″) from the fluid inlet port 4. Theoptical cell 6 in this embodiment is dimensioned to accommodate about 25μL of sample.

Specimen Collection and Preparation

The cartridge may be used to determine the level of microbialcontamination in a fluid, for example, a fluid to be administeredlocally or systemically, for example, parenterally to a mammal, or abody fluid to be tested for infection, including, for example, blood,lymph, urine, serum, plasma, ascites fluid, lung aspirants, and thelike. In addition, the cartridge may be used to determine the level ormicrobial contamination in a water supply, for example, a supply ofdrinking water. In addition, the cartridge may be used to determine thelevel of microbial contamination in a food product, pharmaceutical, ormedical device. Furthermore, the cartridge can be used to determine thelevel of microbial contamination on a surface. For example, the surfaceof interest is swabbed and then the swab is introduced into or dissolvedin liquid. The liquid can then be assayed as usual.

In general, materials used to harvest, store, or otherwise contact asample to be tested, as well as test reagents, should be free ofmicrobial contamination, for example, should be pyrogen-free. Materialsmay be rendered pyrogen-free by, for example, heating at 250° C. for 30minutes. Appropriate precautions should be taken to protectdepyrogenated materials from subsequent environmental contamination.

Representative Assays that can be Performed in the Cartridge

It is contemplated that a variety of hemocyte preparation-based assaysmay be used in the cartridge, such as, for example, an endpointchromogenic assay, a single-step kinetic assay, and a multi-step kineticassay.

1. Endpoint Chromogenic Assay

The endpoint chromogenic assay is described in Prior (1990) supra, pp.28-34, and U.S. Pat. Nos. 4,301,245 and 4,717,658. Briefly, the endpointchromogenic assay includes the steps of (i) solubilizing a hemocytepreparation with a sample to be analyzed, (ii) incubating the resultingmixture at a temperature of about 0° C. to about 40° C., preferablyabout 25° C. to about 40° C., for a predetermined time, (iii) contactinga test device containing substrate, for example, a phenol oxidasesubstrate, with the incubated sample-hemocyte preparation mixture, (iv)adding a reaction inhibitor, and (v) measuring, e.g., by colorimetricchange, a substance produced from the substrate by enzymatic activity.

Referring to FIG. 4A, in order to perform an endpoint chromogenic assayin a cartridge 1, a sample is moved, for example, to a first region 14of the conduit 8 containing the hemocyte preparation, where it issolubilized, for example, by cycling between forward and reverse pumpaction. Following a predetermined incubation period, the sample-hemocytepreparation mixture then is moved, for example, by pump action to asecond region 16 of the conduit 8 containing the phenol oxidasesubstrate, where it is solubilized, for example, by cycling betweenforward and reverse pump action. The sample-hemocytepreparation-substrate mixture then is moved to a third region containinga reaction inhibitor. Afterwards, the sample-hemocytepreparation-substrate mixture is moved to optical cell 6 for measurementof an optical property, for example, the absorbance or transmittanceproperties of the sample by an optical detector. The optical property ofthe sample-hemocyte preparation-substrate mixture at a certainpredetermined time point can then be interpolated onto a predeterminedstandard curve, for example, showing absorbance, optical density, ortransmittance on the Y axis versus lipoteichoic acid concentration onthe X axis, to give the concentration of the microbial contaminant inthe sample.

2. Single-Step Kinetic Assay

A single-step kinetic assay, for example, a single step-chromogenicassay, is described in U.S. Pat. No. 5,310,657. Briefly, the kineticchromogenic assay includes the steps of (i) simultaneously solubilizinga hemocyte preparation with a sample to be analyzed and a substrate, forexample, phenol oxidase substrate, (ii) incubating the resulting mixtureat a temperature of about 0° to about 40° C., preferably about 25° toabout 40° C., over a predetermined time range, and (iii) measuring atime required for a calorimetric change to reach a pre-selected value orchange of the colorimetric readout, using a conventionalspectrophotometer.

Referring to FIG. 4A, in order to perform a kinetic chromogenic assay ina cartridge 1, a sample is moved, for example, by pump action, to afirst region 14 of the conduit 8 containing both the hemocytepreparation and substrate, where it is solubilized, for example, bycycling between forward and reverse pump action. The sample-hemocytepreparation-substrate mixture then is moved to optical cell 6 formeasurement of an optical property, for example, the absorbance ortransmittance properties of the sample by an optical detector. Thedetector may determine how long it takes for each optical property toexhibit, for example, a 5% drop in optical transmittance. Results frommultiple assays, for example, two assays, can be averaged. The resultingvalues may then be interpolated onto a predetermined standard curve, forexample, showing the time for a preselected change in absorbance ortransmittance (as the case may be) on the Y axis versus lipoteichoicacid concentration on the X axis, to give the concentration of thecontaminant in the sample.

3. Multi-Step Kinetic Assay

As will be discussed in more detail, the cartridge may also be used toperform a multi-step kinetic assay. The various steps involved in themulti-step kinetic assay are shown schematically in FIG. 6. The assay isinitiated by combining the sample to be tested with a volume of ahemocyte preparation to produce a sample-hemocyte preparation mixture.The mixture then is incubated for a predetermined period of time. Themixture then is contacted with a substrate, for example, a phenoloxidase substrate, to produce a sample-hemocyte preparation-substratemixture. Thereafter, the time in which a preselected change in anoptical property (for example, a specific change in an absorbance valueor a specific change in a transmission value) is measured. The presenceand/or amount of microbial contaminant may be then determined byinterpolating the measured time against a pre-calibrated standard curve,for example, a standard curve showing the time to make a preselectedchange in optical property (absorbance or transmittance) on the Y axisversus lipoteichoic acid concentration on the X axis.

The standard curve may be created, for example, by adding increasingamounts of an agent, for example, lipoteichoic acid, in a blank sample,for example, pyrogen-free water. The time for which a preselected changein an optical property, for example, a preselected increase inabsorbance or a preselected decrease in transmittance, is determined foreach concentration of lipoteichoic acid. The various time measurementsto achieve a standard change in optical property then are plotted as afunction of the lipoteichoic acid concentration. In general, theconcentration of lipoteichoic acid is inversely proportional to the timenecessary to achieve the standard change in optical property. Thestandard curve can then be used to assess the presence and/or amount oflipoteichoic acid in the sample of interest.

As will be apparent to one skilled in the art, the relative amounts ofhemocyte preparation and phenol oxidase substrate can be adjusted toensure that effective amounts of these two components are present in thesample-hemocyte preparation-substrate mixture at the end of the assay.The final amount of hemocyte preparation protein in the assay is fromabout 1 μg to about 500 μg, preferably about 20 μg. The final amount ofthe substrate, for example, the phenol oxidase substrate in the assay isfrom about 1 μg to about 50 μg, preferably about 6.5 μg. Thedetermination of the concentration and composition of the substrate, forexample, the phenol oxidase substrate, is considered to be within thelevel of skill in the art.

The final volume of the resulting sample-hemocyte preparation-substratemixture can be based on the requirements of the optical detector used tomeasure the change in optical property of the sample. The ratio ofvolumes between the sample, lysate, and substrate can be readilyestablished by those of ordinary skill in the art. Depending on therelative volumes of the sample, hemocyte preparation, and substrate inthe sample-hemocyte preparation-substrate mixture, the concentration ofthe other components of the assay can be adjusted to maintain the finalconcentrations in the operable range, as described herein.

Referring to FIG. 4A, to perform the multi-step kinetic assay in anexemplary cartridge 1, a sample is first moved, for example, by pumpaction, to a first region 14 containing the hemocyte preparation, whereit is mixed and incubated for a predetermined period of time. Thesample-hemocyte preparation mixture then is moved, for example, by pumpaction, to the second region 16 containing the substrate, for example,the phenol oxidase substrate, where it is solubilized. The sample-phenoloxidase-substrate mixture then is moved to optical cell 6, for ameasurement of an optical property. The time intervals required formixing and incubating steps are preprogrammed for optimal sensitivityand microbial contaminant concentration range.

It is understood that a spiked sample can be assayed in parallel with anunspiked sample. The microbial contaminant concentration in the unspikedsample and the microbial contaminant recovered in the spiked sample canbe compared to determine the presence of interference, such as aninhibitor or an enhancer of the reaction, as previously described.

Although the multi-step assay may be performed in a cartridge of thetype discussed above, it may be employed in a variety of other formats,for example, within the well of a microtiter plate. Multiple samples ofvarious test fluids, as well as spiked samples and the series of controlsamples making up a standard curve, may be placed in the wells of themicroplate. Fixed amounts of hemocyte preparation and phenol oxidasesubstrate are added to each of the wells, preferably using an automatedsystem, such as a robot, and the plate processed by a microplate reader,which can be programmed to sequentially read the absorbance of each wellin a repetitive fashion.

EXAMPLES

Practice of the invention will be more fully understood from thefollowing non limiting examples, which are presented herein forillustrative purposes only, and should not be construed as limiting theinvention in any way.

Example 1 Preparation of Lipoteichoic Acid Reactive Material from Cancerborealis

A preparation of lipoteichoic acid reactive material was made fromCancer borealis hemocytes by the following method.

The appearance of the crab was observed and recorded. Centrifuge tubes(50 mL) were labeled and placed on ice. A sterile 18 gauge needle wasconnected to the syringe in a sterile manner, and the syringe was filledwith 5 mL of Bleed Solution (0.45 M sodium chloride, 0.1 M glucose, 30μM sodium citrate, 26 μM citric acid, and 2.5 μM sodium EDTA, pH 4.6).The interior unsclerotized membrane of one of the crab's hind most legswas washed with 70% ethanol and dried with a sterile wipe. The needlewas carefully inserted into the interior unsclerotized membrane of oneof the washed hind legs. The crab was lifted above the needle and thesyringe gently pulled to elicit blood flow. If no blood flowed into thesyringe, the position of the needle was adjusted (e.g., moved deeperinto the membrane). At least 20-60 mL of blood was obtained from eachcrab. When the syringe was full or blood no longer flowed, the needlewas removed from the syringe and the blood gently transferred to acentrifuge tube.

Additional Bleed Solution was added to make a blood:Bleed Solution ratioof 2:1, mixed gently and kept on ice. For example, if 20 mL of blood wascollected, 10 mL of Bleed Solution was added in total (5 mL BleedSolution already added to the syringe plus an additional 5 mL of BleedSolution).

The above method was performed on each crab with a fresh needle, keepingthe same syringe for all crabs but filling the syringe with 5 mL ofBleed Solution with each new crab. All the tubes were centrifuged at3,000 rpm in a RC-3B refrigerated centrifuge (Sorvall, a division ofKendro Laboratories, Asheville, N.C.) at 10° C. for 5 minutes. The lymphsupernatant was discarded or saved in a clean container for furtherextraction. A 50% bleed volume of Wash Solution (0.45 M sodium chloride,0.1 M glucose, 0.1 M cacodylic acid, pH 7.0) was added to the cellpellet (e.g., 10 mL of Wash Solution was added to a pellet obtained from20 mL of blood), and the pellet was gently resuspended using a pipette.The tubes were centrifuged at 3,000 rpm in a RC-3B refrigeratedcentrifuge (Sorvall, a division of Kendro Laboratories, Asheville, N.C.)at 10° C. for 5 minutes. The Wash Solution was discarded and a 10% bleedvolume of Release Solution (0.45 M sodium chloride, 0.1 M glucose, 0.1 Mcacodylic acid, 3 μM calcium ionophore C7522 (Sigma Chemical Co., St.Louis, Mo.), 5 μM calcium chloride, pH 7.0) was added to the pellet(e.g., 2 mL of Release Solution was added to a pellet prepared from 20mL of blood). The pellet was gently resuspended using a pipette. Theresulting solution was vortexed carefully for about 1-2 seconds andincubated for 1 hour at room temperature. The tubes then werecentrifuged at 3,000 rpm in a RC-3B refrigerated centrifuge at 10° C.for 5 minutes and the supernatant was transferred to new tubes fortesting.

Once the pro-phenol oxidase activity of the lysate was determined, theresulting preparation was either used freshly in an assay, lyophilized,dried on a solid support (for example, a cartridge) or aliquoted intotubes (for example, 1-2 mL tubes and frozen at either −20° C. to −80°C.) until use.

Example 2 Protocol for Lipoteichoic Acid Assay

In this assay, a single-step kinetic assay was performed in a microtiterplate. In this assay, lipoteichoic acid (LTA) (L2515, Sigma ChemicalCo., St. Louis, Mo.), was diluted to give a standard curve. The standardcurve contained 3 fold serial dilutions starting from 60 μg/mL to 0.74μg/mL in 0.2 M Tris buffer pH 7.4. Tris buffer (0.2 M, pH 7.4) was usedas a negative control. Comparable standard curves were also createdusing peptidoglycan (77140, Fluka, Switzerland), glucan (glucanstandard, Charles River Endosafe, Charleston, S.C.) andlipopolysaccharide (L-2637, Sigma Chemical Co., St. Louis, Mo.).

The C. borealis preparation used in this Example was preparedessentially as described in Example 1. However, the supernatant prior totesting for pro-phenyl oxidase activity was further purified as follows.Briefly, the supernatant was harvested and an equal volume of asaturated solution of ammonium sulfate was added to the supernatant andmixed gently. Then, the mixture was incubated on ice for 1 hour on ice,and was then centrifuged in a RC-3B refrigerated centrifuge at 10° C.for 5 minutes. The resulting pellet was harvested and resuspended in anamount of distilled water equivalent to the volume of supernatantinitially combined with the saturated ammonium sulfate. The resultingpreparation can then be tested for pro-phenyl oxidase activity.

The lipoteichoic acid, peptidoglycan, glucan and lipopolysaccharidestandards and controls were added to appropriate wells (120 μL perwell). Then, 15 μL of the C. borealis hemocyte preparation was added toeach well. 10 μL of isoprenaline sulfate substrate ((I0261, TCI-GR,Tokyo, Japan) 16 mM in clean water) was then added to each well andgently mixed by shaking for 3-5 seconds. The absorbance of each well wasread periodically (i.e., kinetically) at 490 nm at 37° C. for 40-60minutes (Min OD: 0, Max OD: 0.4, Onset OD: 0.05). The results werereported in optical density units and shown in FIG. 7.

FIG. 7 shows that the Cancer borealis preparation was activated bylipoteichoic acid ranging in concentration from 60 μg/mL to less than2.2 μg/mL. In contrast, peptidoglycan, glucan, and lipopolysaccharidedid not activate the Cancer borealis preparation at the concentrationstested. These results show that, under the appropriate conditions, theCancer borealis preparation can specifically detect lipoteichoic acid.

Example 3 Use of Cancer borealis Hemocyte Preparation for the Detectionof Gram Positive Bacteria

In this Example, a Cancer borealis preparation produced according toExample 1 was used to detect the presence of Gram positive bacteria intest samples. In particular, in this Example, the reactivity of thepreparation was tested against various Gram positive bacteria(including, Staphylococcus epidermidis, Bacillus subtilis, Bacilluspumilus, Bacillus megaterium and Deinococcus radiodurans), Gram negativebacteria (including, Escherichia coli, Pseudomonas stutzeri,Sphingomonas subarctica), Actinomyces (including Nocardiopsisantartica), and fungi (Aureobasidium pullulans).

In this example, the reactivity was assessed using a single-step kineticassay implemented in a microtiter plate. In this assay, lipoteichoicacid (LTA) (L2515, Sigma Chemical Co., St. Louis, Mo.), was diluted togive a standard curve. The standard curve contained 5 fold serialdilutions starting from 100 μg/mL to 0.032 μg/mL in Vasse Media (5.5 mMD-(+)-Glucose, 40 mM Potassium Phosphate (dibasic), 15 mM PotassiumPhosphate (monobasic), 0.4 mM Magnesium Sulfate, 7.5 mM AmmoniumSulfate, 2 mM Citric Acid, Tryptone (10 g/L), pH 7.0). Vasse Media, pH7.0, was used as a negative control. Comparable 10 fold curves were alsocreated using lipopolysaccharide (L-2637, Sigma Chemical Co., St. Louis,Mo.), Staphylococcus epidermidis (Vasse Media, Tryptic Soy Broth),Bacillus subtilis (Vasse Media, Tryptic Soy Broth), Bacillus megaterium(Vasse Media, Tryptic Soy Broth), Bacillus pumilus (Tryptic Soy Broth),Deinococcus radiodurans (Tryptic Soy Broth), Escherichia coli (TrypticSoy Broth), Pseudomonas stutzeri (Tryptic Soy Broth), Sphingomonassubarctica (Tryptic Soy Broth), Nocardiopsis antartica (Tryptic SoyBroth), and Aureobasidium pullulans (Tryptic Soy Broth).

The lipoteichoic acid standard, lipopolysaccharide and bacterial samplesand controls were added to appropriate wells (120 μL per well). Wells ofthe microplate contained 10 μL of dried Cancer borealis hemocytepreparation. Then, 10 μL of isoprenaline sulfate substrate ((I0261,TCI-GR, Tokyo, Japan) 16 mM in clean water) was added to each well. Theabsorbance of each well was read periodically (i.e., kinetically) at 490nm at 37° C. for 90 minutes (Min OD: 0, Max OD: 1.0, Onset OD: 0.1).

The results are reported as the change in optical density as a functionof cell density. The reactivity of the preparation against differentnumbers of cells of the Gram positive organism Staphylococcus epidermis(expressed in optical density units per cell number) is listed in Table1 and shown graphically in FIG. 8. The results show that the Cancerborealis preparation can detect less than 2×10³ cells/mL ofStaphylococcus epidermis. TABLE 1 Cell Number/mL Optical DensityUnits/mL 2 × 10⁷ 1.021 2 × 10⁶ 0.852 2 × 10⁵ 0.146 2 × 10⁴ 0.111 2 × 10³0.096

The reactivity of the preparation against different numbers of cells ofthe Gram positive organism, Bacillus subtilis, (expressed in opticaldensity units per cell number) is listed in Table 2, and showngraphically in FIG. 9. The results show that the Cancer borealispreparation can detect between 3×10⁵ and 6×10⁴ cells/mL of Bacillussubtilis. TABLE 2 Cell Number/mL Optical Density Units/mL 4 × 10⁷ 22.668 × 10⁶ 14.96 2 × 10⁶ 2.97 3 × 10⁵ 0.3 6 × 10⁴ 0 Control 0

The reactivity of the Cancer borealis preparation against a number ofGram positive bacteria, Gram negative bacteria, fungi, and actinomycetesis summarized in Table 3.

The results show that the Cancer borealis preparation detected all ofthe Gram positive organisms. In contrast, the Cancer borealispreparation did not detect either the Gram negative bacteria or thefungus Aureobasidium pullulans. Although, the Cancer borealispreparation detected the presence of the actinomycetes, Nocardiopsisantartica, this organism stains like the Gram positive bacteria. TABLE 3Cell Optical Concen- Density Organism Name Type of Organism trationUnits Bacillus subtilis Gram Positive bacteria   2 × 10⁶ 1.188 Bacilluspumilus Gram Positive bacteria 4.7 × 10⁷ 2.238 Bacillus megaterium GramPositive bacteria 3.5 × 10⁷ 2.577 Bacillus species Gram Positivebacteria   3 × 10⁶ 1.053 Deinococcus Gram Positive bacteria 2.1 × 10⁶2.019 radiodurans Escherichia coli Gram Negative bacteria   1 × 10⁹ 0Pseudomonas stutzeri Gram Negative bacteria 2.9 × 10⁸ 0 SphingomonasGram Negative bacteria 1.9 × 10⁷ 0 subarctica Nocardiopsis antarticaActinomyces   1 × 10⁵ 1.453 Aureobasidium Fungi 4.8 × 10⁶ 0 pullulans

Example 4 Preparation of Lipoteichoic Acid Reactive Material fromLimulus polyphemus

This Example demonstrates that Limulus polyphemus hemocytes containlipoteichoic acid reactive material capable of detecting the presence ofGram positive bacteria. Blood was collected from Limulus polyphemus andthe lipoteichoic acid reactive material was produced as described inExample 1.

The resulting preparation then was tested for reactivity againstlipoteichoic acid, lipopolysaccharide and glucan. This assay was atwo-step kinetic assay, performed in a microtiter plate. In this assay,lipoteichoic acid (LTA) (L2515, Sigma Chemical Co., St. Louis, Mo.), wasdiluted to give a standard curve. The standard curve contained 10 foldserial dilutions starting from 100 μg/mL to 0.01 μg/mL in 0.2 M Trisbuffer pH 7.4. Tris buffer (0.2 M, pH 7.4) was used as a negativecontrol. Comparable standard curves were also created using glucan(glucan standard, Charles River Endosafe, Charleston, S.C.) andlipopolysaccharide (L-2637, Sigma Chemical Co., St. Louis, Mo.).

The lipoteichoic acid, peptidoglycan, glucan, lipopolysaccharidestandards and controls then were added to the appropriate wells (100 μLper well). Then, 10 μL of Limulus hemocyte preparation was added to eachwell and gently mixed by shaking for 3-5 seconds. Then, the microplatewas incubated at 37° C. for 20 minutes. Then, 10 μL of isoprenalinesulfate substrate ((10261, TCI-GR, Tokyo, Japan) 4 mM in clean water)was added to each well and gently mixed by shaking for 3-5 seconds. Theabsorbance of each well was read periodically (i.e., kinetically) at 490nm at 37° C. for 90 minutes (Min OD: 0, Max OD: 0.4, Onset OD: 0.05).The results expressed as a change in optical density (+) or no change inoptical density (−) are summarized in Table 4. These results demonstratethat the Limulus preparation is capable of detecting lipoteichoic acid.The results also demonstrate that the Limulus preparation is alsocapable of detecting lipopolysaccharide and glucan. TABLE 4Concentration Lipoteichoic acid Lipopolysaccharide Glucan 100μg/ml + + + 10 μg/ml + + + 1 μg/ml + − − 100 ng/ml − − − 10 ng/ml − − −Control − − −

Example 5 Preparation of Cartridge for Use with Cancer borealisPreparation in a Two-Step Kinetic Assay

In this Example, a Cancer borealis preparation from Example 1 wasincorporated into a cartridge and used in a two-step kinetic assay. Thereactivity of the C. borealis preparation to different concentrations oflipoteichoic acid (LTA) (L2515, Sigma Chemical Co., St. Louis, Mo.) (0μg/mL, 5.6 μg/mL, 16.7 μg/mL and 50 μg/mL) was tested in the cartridgeusing The Endosafe—PTS (Portable Test System) (Charles RiverLaboratories, Charleston, S.C.).

Cancer borealis hemocyte preparation was dried in the cartridge asdescribed above in the Cartridge Fabrication section. Mannitol (1%) andpolyvinyl alcohol (0.1%) were added to a dilute form of Cancer borealishemocyte preparation (diluted 1:2 in pyrogen free water) and then 5 μLof this solution was placed in the appropriate location in the cartridgefor drying.

A cartridge of the type shown in FIG. 4 was prepared as follows.Referring to FIG. 5A, the Cancer borealis preparation and isoprenalinesulfate substrate was applied to regions 14′ and 16′, respectively, ofconduit 8′ of the bottom half 2 of the cartridge 1 using a HamiltonMicrolab 540B Dispenser (Hamilton Company, Reno, Nev.). Briefly, 5 μL ofa dilute Cancer borealis preparation (1:2 in pyrogen free water withmannitol (1%) and polyvinyl alcohol (0.1%)) was applied to region 14′. 5μL of isoprenaline sulfate substrate ((10261, TCI-GR, Tokyo, Japan) 6 mMin clean water, 1% Mannitol and 0.1% polyvinyl alcohol) was applied toregion 16′. The bottom half 2 of the cartridge 1 was dried under acontrolled temperature of 25° C.+/−2° C. and a humidity of 5%+/−5% in aLunaire Environmental Steady State & Stability Test Chamber (LunaireEnvironmental, Williamsport, Pa.) in a Puregas HF200 Heatless Dryer (MTIPuregas, Denver, Colo.) for 1 hour. Temperature and humidity wascontrolled by a Watlow Series 96 1/16 DIN Temperature Controller (WatlowElectric Manufacturing Company, St. Louis, Mo.).

Referring to FIG. 5B, a top half of the cartridge was prepared without aspike (lipoteichoic acid) applied to region 14″ of the conduit 8″ of thetop half 3 of the cartridge 1. Following fabrication, the two halves 2and 3 were assembled such that regions 14′ and 14″ were aligned one ontop of the other, and the edges of the cartridge halves 2 and 3 wereultrasonically sealed using a Dukane Model 210 Ultrasonic Sealer (DukaneCorporation, St. Charles, Ill.) under the control of a Dukane DynamicProcess Controller (Dukane Corporation, St. Charles, Ill.).

The results are summarized in FIG. 10 and show that the Cancer borealispreparation has no reactivity in the absence of lipoteichoic acid. Thereactivity of the preparation, however, increased the presence ofincreasing concentrations of lipoteichoic acid. These resultsdemonstrate that the Cancer borealis preparation is capable of detectinglipoteichoic acid present in or on Gram positive bacteria.

Example 6 A Hemocyte-Based Preparation Capable of Detecting GramPositive Bacteria, Gram Negative Bacteria and Fungi and Molds

The Cancer borealis preparation made in accordance with the teachings ofExample 1 is capable of detecting the presence of Gram positive bacteriabut not Gram negative bacteria, yeasts and molds. To the extent that apreparation capable of detecting Gram positive bacteria, Gram negativebacteria, and yeasts and molds is desirable, it is possible to createsuch a preparation by combining the Cancer borealis preparation ofExample 1 (reactive with Gram positive bacteria) with crude Limulusamebocyte lysate (reactive with Gram negative bacteria and yeast andmolds).

Briefly, a crude Limulus amebocyte lysate prepared as originallydescribed in Levin et al. ((1968) THROMB. DIATH. HAEMORRH. 19: 186) wascombined in an equal amount with the Cancer borealis preparationprepared according to Example 1.

In this assay, a single-step kinetic assay was performed in a microtiterplate and lipoteichoic acid (LTA) (L2515, Sigma Chemical Co., St. Louis,Mo.) was diluted to give a standard curve. The standard curve contained5 fold serial dilutions starting from 100 μg/mL to 1.28 ng/mL in Trisbuffer (0.2 M, pH 7.4). Tris buffer (0.2M, pH 7.4) was used as anegative control. Comparable 5 fold curves were also created usingpeptidoglycan (77140, Fluka, Switzerland), glucan (glucan standard,Charles River Endosafe, Charleston, S.C.) and lipopolysaccharide(L-2637, Sigma Chemical Co., St. Louis, Mo.).

The lipoteichoic acid standard, peptidoglycan, glucan,lipopolysaccharide and controls then were added to the appropriate wells(120 μL per well). Wells of the microplate contained 5 μL of dried C.borealis hemocyte preparation. 5 μL of crude Limulus amebocyte lysatepreparation then was added to the wells. Finally, 10 μL of isoprenalinesulfate substrate ((10261, TCI-GR, Tokyo, Japan) 16 mM in clean water)was added to each well. The absorbance of each well was readperiodically (i.e., kinetically) at 490 nm at 37° C. for 150 minutes(Min OD: 0, Max OD: 1.5, Onset OD: 0.2). The results are summarized inFIG. 11 and show that the preparation reacts with lipopolysaccharide,peptidoglycan, glucan and lipoteichoic acid. These data suggest thatsuch lysates can detect Gram positive bacteria, Gram negative bacteriaand yeasts and fungi.

Example 7 Lipoteichoic Assays Using Fluorescent Substrates for PhenolOxidase

This Example shows that a fluorescent phenol oxidase substrate can beused in a single-step kinetic assay in a well type format.

The assay was performed as follows. Cancer borealis lipoteichoic acidreactive material was produced as described in Example 1. 10 μL of theCancer borealis preparation (1% mannitol) was dried into each well of amicrotiter plate. Serial dilutions of lipoteichoic acid were prepared in0.2 M Tris buffer (pH 7.4). Then, 120 μL of the lipoteichoic acidstandards were added to the wells to give a pair of wells having a finalconcentration of 100 μg/mL, 50 μg/mL, 25 μg/mL, and 12.5 μg/mL. Thecontrol only contained 120 μL of Tris buffer. The plate was incubated at37° C. for 30 minutes. Then, 20 μL of 2′,7′-dichlorodihydrofluoresceindiacetate (DCFDA) (D-399, Molecular probes, Eugene, Oreg.) and 8 mMcatechol (C-9510, Sigma Chemical Co., St. Louis, Mo.) was added to onewell of the pair. 20 μL of DCFDA, 8 mM catechol and 1/250 dilution ofesterase (E-2884, Sigma Chemical Co., St. Louis, Mo.) was added to onewell of the pair of wells. Absorbance at 490 nm was recorded for 120minutes. The results, expressed in fluorescence units versusconcentration of lipoteichoic acid, are presented in FIG. 12.

FIG. 12 shows that a fluorescent substrate can be used in an assay. Theresults show that although the assay can work in the presence andabsence of the esterase, the assay is more sensitive in the presence ofesterase. For example, 100 μg/mL of lipoteichoic gives a signalequivalent to about 1 fluorescence unit in the absence of esterase and asignal equivalent to about 4.8 fluorescence units in the presence ofesterase. This type of assay may be useful with colored test solutions,for example, blood samples.

Example 8 Lipoteichoic Acid Reactive Material in Crude Limulus AmebocyteLysate

Example 4 shows that lipoteichoic acid reactive material can beharvested from calcium ionophore permeabilized Limulus polyphemushemocytes. This example demonstrates that lipoteichoic acid reactivematerial is also present in crude Limulus polyphemus hemocyte lysate.

Briefly, Limulus amebocyte lysate was prepared as originally describedin Levin et al. ((1968) supra). The lysate was assayed for the presenceof lipoteichoic acid reactive material as described in Example 2. Theresults indicate that crude Limulus amebocyte lysate can detect thepresence of lipoteichoic acid in a sample. It is interesting that as theconcentration of the crude lysate in the sample mixture is decreased,the signal increased. These results suggest that crude Limulus amebocytelysate contains an inhibitor for the pro-phenol oxidase cascade.However, it appears that the inhibitor can be titrated out so that thecrude lysate can detect the presence of lipoteichoic acid in a sample.Accordingly, it appears that the crude Limulus amebocyte lysate candetect Gram positive bacteria. Furthermore, it appears that the crudelysate, under the appropriate conditions can detect the presence of Grampositive bacteria (via the pro-phenol oxidase cascade), Gram negativebacteria (via the Factor C cascade) and yeasts and molds (via the FactorG cascade).

EQUIVALENTS

The invention may be embodied in other specific forms without departingfrom the spirit or essential characteristics thereof. The foregoingembodiments are therefore to be considered in all respects illustrativerather than limiting on the invention described herein. Scope of theinvention is thus indicated by the appended claims rather than by theforegoing description, and all changes that come within the meaning andrange of equivalency of the claims are intended to be embraced therein.

INCORPORATION BY REFERENCE

All publications and patent documents cited in this application areincorporated by reference in their entirety for all purposes to the sameextent as if the entire contents of each individual publication orpatent document was incorporated herein.

1. A method of producing a preparation for detecting the presence and/oramount of lipoteichoic acid in a sample, the method comprising the stepsof: (a) providing a hemocyte preparation harvested from a crustaceanselected from the group consisting of Cancer borealis, Cancer irroratus,Hemigrapsus sanguineus, and Limulus polyphemus, wherein the hemocytescontain lipoteichoic acid reactive material disposed therein; (b)releasing the lipoteichoic acid reactive material from the hemocytes;and (c) harvesting the lipoteichoic reactive material released from thehemocytes.
 2. The method of claim 1, wherein in step (b) thelipoteichoic acid reactive material is released by lysis of thehemocytes.
 3. The method of claim 1, wherein in step (b) thelipoteichoic acid reactive material is released when the hemocytes arerendered permeable to lipoteichoic acid reactive.
 4. The method of claim3, wherein the hemocytes are rendered permeable by exposure to amembrane permeabilizing agent.
 5. The method of claim 4, wherein thepermeabilizing agent is an ionophore.
 6. The method of claim 5, whereinthe ionophore is a calcium ionophore.
 7. The method of claim 5, whereinthe ionophore is admixed with the hemocyte preparation to give a finalconcentration in the range from about 0.1 μM to about 100 μM.
 8. Themethod of claim 7, wherein the ionophore is admixed with the hemocytepreparation to give a final concentration in the range from about 1 μMto about 10 μM.
 9. The method of claim 5, wherein the membrane isrendered permeable by exposing the membrane to the ionophore in thepresence of divalent cations.
 10. The method of claim 9, wherein thecations are calcium ions.
 11. The method of claim 1, wherein thecrustacean is Limulus polyphemus.
 12. The method of claim 1, wherein thelipoteichoic acid reactive material comprises pro-phenol oxidase. 13.The method of claim 1, wherein the lipoteichoic acid reactive materialcomprises an enzyme, which is not activated when released from thehemocytes but when later activated converts pro-phenol oxidase intophenol oxidase.
 14. The method of claim 1, comprising an additional stepof, after step (c), combining the harvested lipoteichoic reactivematerial with a substrate for phenol oxidase.
 15. A method of producinga preparation for detecting the presence and/or quantity of lipoteichoicacid in a sample, the method comprising the steps of: (a) providing ahemocyte preparation, wherein hemocytes in the preparation (i) have anouter membrane and (ii) contain lipoteichoic acid reactive materialdisposed therein; (b) rendering the outer membrane of at least a portionof the hemocytes in the hemocyte preparation permeable to thelipotechoic acid reactive material so that the lipoteichoic acidreactive material is released from the hemocytes; and (c) harvesting thelipoteichoic acid reactive material released from the hemocytes.
 16. Themethod of claim 15, wherein in step (b), the outer membrane is renderedselectively permeable by exposure to a membrane permeabilizing agent.17. The method of claim 16, wherein the permeabilizing agent is anionophore.
 18. The method of claim 17, wherein the ionophore is acalcium ionophore. 19-22. (canceled)
 23. The method of claim 15, whereinthe hemocytes are derived from a crustacean.
 24. The method of claim 23,wherein the crustacean is selected from the group consisting of Cancerborealis, Cancer irroratus, Carcinus maenas, Hemigrapsus sanguineus, andLimulus polyphemus.
 25. The method of claim 23, wherein the crustaceanis Cancer borealis or Limulus polyphemus.
 26. The method of claim 15,wherein the lipoteichoic acid reactive material comprises pro-phenoloxidase.
 27. The method of claim 15, wherein the lipoteichoic acidreactive material comprises an enzyme, which when activated convertspro-phenol oxidase into phenol oxidase.
 28. The method of claim 15comprising the additional step of, after step (c), combining theharvested lipoteichoic acid reactive material with a substrate forphenol oxidase.
 29. A method of producing a preparation of lipoteichoicacid reactive material, the method comprising the steps of: (a)providing a hemocyte preparation, wherein hemocytes in the preparation(i) have an outer membrane and (ii) contain lipoteichoic acid reactivematerial disposed therein; (b) admixing the hemocytes with a membranepermeabilizing agent to release the lipoteichoic acid reactive materialfrom at least a portion of the hemocytes; and (c) harvesting thelipoteichoic acid reactive material released from the hemocytes.
 30. Themethod of claim 29, wherein the permeabilizing agent makes the outermembrane selectively permeable to the lipoteichoic acid reactivematerial.
 31. The method of claim 30, wherein the permeabilizing agentis an ionophore.
 32. The method of claim 31, wherein the ionophore is acalcium ionophore. 33-36. (canceled)
 37. The method of claim 29, whereinthe hemocytes are derived from a crustacean. 38-39. (canceled)
 40. Themethod of claim 29, wherein the lipoteichoic acid reactive materialcomprises pro-phenol oxidase.
 41. The method of claim 29, wherein thelipoteichoic acid reactive material comprises an enzyme, which whenactivated converts pro-phenol oxidase into phenol oxidase.
 42. Themethod of claim 29 comprising the additional step of, after step (c),combining the harvested lipoteichoic acid reactive material with asubstrate for phenol oxidase. 43-55. (canceled)
 56. A preparation oflipoteichoic acid reactive material produced by the method of claim 1.57-59. (canceled)
 60. A composition comprising an isolated enzymepreparation derived from crustacean hemocytes, wherein the enzymepreparation comprises pro-phenol oxidase which is converted into phenoloxidase when the enzyme preparation is contacted with lipoteichoic acid.61. The composition of claim 60, wherein the enzyme preparation isderived from hemocytes whose outer membranes have been renderedselectively permeable to pro-phenol oxidase.
 62. (canceled)
 63. Thecomposition of claim 60, wherein the crustacean is Cancer borealis. 64.The composition of claim 60, wherein the enzyme preparation issubstantially free of Factor C activity, Factor G activity, or bothFactor C and Factor G activity.
 65. The composition of claim 60, whereinthe enzyme preparation remains substantially inactivated in the presenceof (1→3)-β-D glucan, lipopolysaccharide, or proteoglycan.
 66. (canceled)67. A crustacean hemocyte lysate comprising pro-phenol oxidase, which isconverted into phenol oxidase when the hemocyte lysate is contacted withlipoteichoic acid.
 68. (canceled)
 69. The composition of claim 60further comprising a substrate for phenol oxidase.
 70. A method ofdetecting the presence of Gram-positive bacteria in a sample, the methodcomprising contacting the sample with the preparation of claim 56.71-72. (canceled)
 73. A method of detecting the presence ofGram-positive bacteria in a sample, the method comprising contacting thesample with the composition of claim
 60. 74-75. (canceled)